The Effect of an Intervening Promoter Nucleosome on Gene Expression

<div><p>Nucleosomes, which are the basic packaging units of chromatin, are stably positioned in promoters upstream of most stress-inducible genes. These promoter nucleosomes are generally thought to repress gene expression due to exclusion; they prevent transcription factors from accessing their target sites on the DNA. However, the role of promoter nucleosomes that do not directly occlude transcription factor binding sites is not obvious. Here, we varied the stability of a non-occluding nucleosome positioned between a transcription factor binding site and the TATA box region in an inducible yeast promoter and measured downstream gene expression level. We found that gene expression level depends on the occupancy of the non-occluding nucleosome in a non-monotonic manner. We postulated that a non-occluding nucleosome can serve both as a vehicle of and a barrier to chromatin remodeling activity and built a quantitative, nonequilibrium model to explain the observed nontrivial effect of the intervening nucleosome. Our work sheds light on the dual role of nucleosome as a repressor and an activator and expands the standard model of gene expression to include irreversible promoter chromatin transitions.</p></div

Gene regulation functions of Nuc -2 (+) and Nuc -2 (−) variants.

<p>To generate the GRF, data points were binned by their YFP (input) intensity values, and the mean and standard deviation of CFP (output) intensity values within each bin were obtained. The mean and standard deviation from each bin were averaged from three independent measurements. Double-averaged CFP intensities are shown as circles, and the averaged standard deviations are indicated by the vertical width of the shaded region. The error bar represents the standard error of the mean. Nuc -2 (<b>−</b>) variant is shown in grey, and 24%, 39%, and 54% Nuc -2 (+) variants are shown in green, blue, and red, respectively. The GRFs of Nuc -2 (+) variants are fitted to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063072#pone.0063072.e001" target="_blank">Equation 1</a>. p₁ = 10^2.4, p₂ = 10^3.6, p₄ = 0.97, and p₃ is 99, 703, and 363 for 24, 39, and 54 GC%, respectively.</p

Chromatin architecture of the promoter variants used in this study.

<p>DNA is shown as a solid black line. Nucleosomes -1, -2 and -3 are numbered relative to the transcription start site (TSS) and shown in dark ellipses. The direction of transcription is indicated by the black bent arrow. The TATA box is marked in nucleosome -1 (solid yellow tick). A high affinity Pho4 binding site (CACGTG) is located in the linker region between nucleosome -3 and nucleosome -2 (solid purple tick). Nucleosome -2 is the non-occluding nucleosome. Three Nuc -2 (+) variants are used in this study: low (24%), intermediate (39%), and high (54%) GC% DNA sequence for the non-occluding nucleosome. This DNA segment is deleted in Nuc -2 (−) promoter variant.</p

In-vivo nucleosome occupancy maps of Nuc -2 (+) variants.

<p>Relative nucleosome occupancies (in arbitrary units) were measured by quantitative ChIP at five locations (in base pairs) which are near or in between the putative centers of promoter nucleosomes according to the compiled data base for nucleosome positioning in <i>S. cerevisiae </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063072#pone.0063072-Jiang1" target="_blank">[33]</a>. The nucleosome occupancy at REC104 locus was used as the reference value. The error bars represent the standard deviation of three separate ChIP measurements starting with independent sample preparation.</p

Modeling the effect of the intervening promoter nucleosome on gene expression.

<p>(A) The proposed model for nucleosome removal by the chromatin remodeling complex. The promoter is in dynamic equilibrium between two states based on the occupancy of nucleosome -2. The chromatin remodeling complex SWI/SNF is recruited by the transcription factor Pho4 to the linker region. If nucleosome -2 is present (transcriptionally active path), SWI/SNF can bind it and begin pulling DNA within nucleosome -2 from nucleosome -1. This results in collision between nucleosome -1 and the head of SWI/SNF, and the eventual removal of nucleosome -1. If nucleosome -2, however, is not present (transcriptionally inactive path), SWI/SNF cannot easily reach the next nearest nucleosome -1 because of ∼200 bp distance. Participating molecules are drawn approximately to scale. (B) Near-equilibrium model of gene expression. The letters, T, C, and N represent transcription factor, nucleosome, and chromatin remodeling complex, respectively. The promoter state is defined by the occupancy of T, C, and N. The first square represents the transcription factor binding site, and the second square the position for the non-occluding nucleosome. <i>K_DT is</i> the equilibrium dissociation constant for T⋅DNA T+DNA, and <i>K_DN</i> for N⋅DNA N+DNA. <i>K_DN</i> represents nucleosome stability and varies among Nuc -2 (+) variants. The irreversible chromatin remodeling step represented by <i>k_r</i> brings (1,1,1) back to (1,0,0), and completes the steady-state cycle.</p

Extracellular synthesis of crystalline silver nanoparticles and molecular evidence of silver resistance from Morganella sp.: towards understanding biochemical synthesis mechanism

There has been significant progress in the biological synthesis of nanomaterials. However, the molecular mechanism of synthesis of such bio-nanomaterials remains largely unknown. Here, we report the extracellular synthesis of crystalline silver nanoparticles (AgNPs) by using Morganella sp., and show molecular evidence of silver resistance by elucidating the synthesis mechanism. The AgNPs were 20±5 nm in diameter and were highly stable at room temperature. The kinetics of AgNPs formation was investigated. Detectable particles were formed after an hour of reaction, and their production remained exponential up to 18 h, and saturated at 24 h. Morganella sp. was found to be highly resistant to silver cations and was able to grow in the presence of more than 0.5 mM AgNO3. Three gene homologues viz. silE, silP and silS were identified in silver-resistant Morganella sp. The homologue of silE from Morganella sp. showed 99% nucleotide sequence similarity with the previously reported gene, silE, which encodes a periplasmic silver-binding protein. The homologues of silP and silS were also highly similar to previously reported sequences. Similar activity was totally absent in closely related Escherichia coli; this suggests that a unique mechanism of extracellular AgNPs synthesis is associated with silver-resistant Morganella sp. The molecular mechanism of silver resistance and its gene products might have a key role to play in the overall synthesis process of AgNPs by Morganella sp. An understanding of such biochemical mechanisms at the molecular level might help in developing an ecologically friendly and cost-effective protocol for microbial AgNPs synthesis

Selected area electron diffraction (SAED) patterns obtained from AgNPs formed by (A) <i>M. morganii</i> strain RP-42, and (B) <i>M. psychrotolerans</i>.

<p>Selected area electron diffraction (SAED) patterns obtained from AgNPs formed by (A) <i>M. morganii</i> strain RP-42, and (B) <i>M. psychrotolerans</i>.</p